Biomaterial Database

DNA sequencing with [alpha-33P]-labeled ddNTP terminators: a new approach to DNA sequencing with Thermo Sequenase DNA polymerase. 1996

J Fan, and R S Ranu, and C Smith, and C Ruan, and C W Fuller

Colorado State University, Fort Collins, USA.

A new approach to DNA sequencing is described. The method is based on the use of [alpha-33P]-labeled dideoxyribonucleoside triphosphate terminators and Thermo Sequenase DNA polymerase in cycle sequencing. Thermo Sequenase DNA polymerase incorporates ddNTPs as efficiently as dNTPs, allowing the use of low concentrations of these nucleotides in DNA sequencing. Because only the properly terminated chains are labeled and visualized on autoradiography of the sequencing gels, the sequence results are free of background. The intensity of DNA bands generated are remarkably uniform, which makes reading of DNA sequences easy. By staggered loading of the sequencing gel (at 2-3 hour intervals), it is possible to sequence DNA at least 450 to 500 nucleotides. Exposure time for autoradiography with [alpha-33P] labels is much shorter than with [35S] and does not substantially compromise autoradiographic resolution. Data can be obtained after only 12 hours of exposure of an X-ray film. Moreover, cycle sequencing requires very small amounts of single- or double-stranded template. Consequently, it is even possible to generate sequence data from a single bacterial colony. The details of the protocol are presented in a stepwise manner, and some important parameters to be considered for sequencing with this method are discussed.

UI	MeSH Term	Description	Entries
D011933	Reagent Kits, Diagnostic	Commercially prepared reagent sets, with accessory devices, containing all of the major components and literature necessary to perform one or more designated diagnostic tests or procedures. They may be for laboratory or personal use.	Diagnostic Reagent Kits,Diagnostic Reagents and Test Kits,Diagnostic Test Kits,In Vitro Diagnostic Device,In Vitro Diagnostic Devices,In Vitro Diagnostic Medical Device,In Vitro Diagnostic Medical Devices,Kits, Diagnostic Reagent,Diagnostic Reagent Kit,Diagnostic Test Kit,Kit, Diagnostic Reagent,Kit, Diagnostic Test,Kits, Diagnostic Test,Reagent Kit, Diagnostic,Test Kit, Diagnostic,Test Kits, Diagnostic
D004259	DNA-Directed DNA Polymerase	DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair.	DNA Polymerase,DNA Polymerases,DNA-Dependent DNA Polymerases,DNA Polymerase N3,DNA Dependent DNA Polymerases,DNA Directed DNA Polymerase,DNA Polymerase, DNA-Directed,DNA Polymerases, DNA-Dependent,Polymerase N3, DNA,Polymerase, DNA,Polymerase, DNA-Directed DNA,Polymerases, DNA,Polymerases, DNA-Dependent DNA
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D017422	Sequence Analysis, DNA	A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis.	DNA Sequence Analysis,Sequence Determination, DNA,Analysis, DNA Sequence,DNA Sequence Determination,DNA Sequence Determinations,DNA Sequencing,Determination, DNA Sequence,Determinations, DNA Sequence,Sequence Determinations, DNA,Analyses, DNA Sequence,DNA Sequence Analyses,Sequence Analyses, DNA,Sequencing, DNA
D017931	DNA Primers	Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques.	DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D018388	Codon, Terminator	Any codon that signals the termination of genetic translation (TRANSLATION, GENETIC). PEPTIDE TERMINATION FACTORS bind to the stop codon and trigger the hydrolysis of the aminoacyl bond connecting the completed polypeptide to the tRNA. Terminator codons do not specify amino acids.	Amber Stop Codon,Codon, Amber Stop,Codon, Ochre Stop,Codon, Opal Stop,Codon, Stop,Stop Codon,Terminator Codon,Amber Codon,Amber Terminator Codon,Codon, Termination,Ochre Codon,Ochre Stop Codon,Opal Codon,Opal Stop Codon,Stop Codon UAA,Stop Codon UAG,Stop Codon UGA,Stop Signal, Translation,TAA Codon,TAG Codon,TGA Codon,UAA Codon,UAA Stop Codon,UAG Codon,UAG Stop Codon,UGA Codon,UGA Stop Codon,Amber Codons,Amber Stop Codons,Amber Terminator Codons,Codon UAA, Stop,Codon UAG, Stop,Codon UGA, Stop,Codon, Amber,Codon, Amber Terminator,Codon, Ochre,Codon, Opal,Codon, TAA,Codon, TAG,Codon, TGA,Codon, UAA,Codon, UAA Stop,Codon, UAG,Codon, UAG Stop,Codon, UGA,Codon, UGA Stop,Codons, Amber,Codons, Amber Stop,Codons, Amber Terminator,Codons, Ochre,Codons, Ochre Stop,Codons, Opal,Codons, Opal Stop,Codons, Stop,Codons, TAA,Codons, TAG,Codons, TGA,Codons, Termination,Codons, Terminator,Codons, UAA,Codons, UAA Stop,Codons, UAG,Codons, UAG Stop,Codons, UGA,Codons, UGA Stop,Ochre Codons,Ochre Stop Codons,Opal Codons,Opal Stop Codons,Stop Codon UGAs,Stop Codon, Amber,Stop Codon, Ochre,Stop Codon, Opal,Stop Codon, UAA,Stop Codon, UAG,Stop Codon, UGA,Stop Codons,Stop Codons, Amber,Stop Codons, Ochre,Stop Codons, Opal,Stop Codons, UAA,Stop Codons, UAG,Stop Codons, UGA,Stop Signals, Translation,TAA Codons,TAG Codons,TGA Codons,Termination Codon,Termination Codons,Terminator Codon, Amber,Terminator Codons,Terminator Codons, Amber,Translation Stop Signal,Translation Stop Signals,UAA Codons,UAA Stop Codons,UAA, Stop Codon,UAG Codons,UAG Stop Codons,UAG, Stop Codon,UGA Codons,UGA Stop Codons,UGA, Stop Codon